With a Novel BCOR Mutation and Genomic Rearrangements Involving NHS

ORIGINAL ARTICLE

A family of oculofaciocardiodental syndrome (OFCD) with a novel BCOR mutation and genomic rearrangements involving NHS

Yukiko Kondo1, Hirotomo Saitsu1, Toshinobu Miyamoto2, Kiyomi Nishiyama1, Yoshinori Tsurusaki1, Hiroshi Doi1, Noriko Miyake1, Na-Kyung Ryoo3, Jeong Hun Kim3, Young Suk Yu3 and Naomichi Matsumoto1

Oculofaciocardiodental syndrome (OFCD) is an X-linked dominant disorder associated with male lethality, presenting with congenital cataract, dysmorphic face, dental abnormalities and septal heart defects. Mutations in BCOR (encoding BCL-6- interacting corepressor) cause OFCD. Here, we report on a Korean family with common features of OFCD including bilateral 2nd–3rd toe syndactyly and septal heart defects in three affected females (mother and two daughters). Through the mutation screening and copy number analysis using genomic microarray, we identiﬁed a novel heterozygous mutation, c.888delG, in the BCOR gene and two interstitial microduplications at Xp22.2–22.13 and Xp21.3 in all the three affected females. The BCOR mutation may lead to a premature stop codon (p.N297IfsX80). The duplication at Xp22.2–22.13 involved the NHS gene causative for Nance–Horan syndrome, which is an X-linked disorder showing similar clinical features with OFCD in affected males, and in carrier females with milder presentation. Considering the presence of bilateral 2nd–3rd toe syndactyly and septal heart defects, which is unique to OFCD, the mutation in BCOR is likely to be the major determinant for the phenotypes in this family. Journal of Human Genetics (2012) 57, 197–201; doi:10.1038/jhg.2012.4; published online 2 February 2012

Keywords: BCOR; congenital cataract; frameshift mutation; genomic rearrangement; Nance-Horan syndrome; NHS; oculofaciocardiodental syndrome

INTRODUCTION site mutations, suggesting that the loss of functions (null allele) might Oculofaciocardiodental syndrome (OFCD, Mendelian Inheritance in result in OFCD. In addition, microdeletions involving BCOR have Man (MIM) #300166), an X-linked dominant disorder, is character- been also reported in individuals with OFCD. Most mutations ized by ocular, facial, cardiac and dental abnormalities associated with predicted to generate premature stop codons, likely suffering from male lethality.1,2 Mutations in the BCL-6 corepressor gene (BCOR, nonsense-mediated mRNA decay, although nonsense-mediated MIM *300485) at Xp11.4 cause OFCD.3 BCOR/Bcor is ubiquitously mRNA decay was unable to be conﬁrmed because of the severe skewed expressed in human tissues and is strongly and speciﬁcally expressed X-inactivation in blood leukocytes.3,6 in the eye, brain, neural tube and branchial arches during mouse Nance–Horan syndrome (NHS) is an X-linked cataract-dental embryonic development, which are affected in OFCD.4,5 In 2009, syndrome (MIM #302350) characterized by congenital cataract, dental Hilton et al.6 clinically reviewed 35 cases with BCOR mutations and abnormalities, facial dysmorphism and mental retardation.7 Conge- summarized the frequency of phenotypes: congenital cataract (100%), nital cataract in affected male usually requires early surgery.8 Dental microphthalmia and/or microcornea (82%), facial dysmorphism abnormalities include maxillary and mandibular diastema of both (96%) including long narrow face and high nasal bridge, cardiac central and lateral incisors, and screwdriver-shaped teeth because of anomalies (74%, commonly septal defects), dental abnormalities narrow gingival and incisal margins.9 Carrier females typically display (100%) such as delayed and/or primary dentition, root radiculome- posterior Y-sutural lens opacities, and the dental and facial anomalies galy, and absent/duplicated/fused teeth and mental retardation of the syndrome may be observed, but with a milder presentation.8 (18%).6 Additionally, skeletal abnormalities such as 2nd–3rd toe Mutations in NHS (MIM *300457) at Xp21.1-p22.3 cause NHS.9–11 syndactyly, hammer toes, and radioulnar synostosis are also observed The most pathogenic mutations are truncating mutations. Coccia in 97% patients. Various types of mutations in BCOR have been et al.8 reported complex duplication-triplication rearrangements described including nonsense, small insertions or deletions and splice of the NHS gene in a family with congenital cataract and congenital

1Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan; 2Department of Obstetrics and Gynecology, Asahikawa Medical College, Asahikawa, Japan and 3Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea Correspondence: Dr N Matsumoto, Department of Human Genetics, Yokohama City University Graduate School of Medicine, Fukuura 3-9, Kanazawa-ku, Yokohama 236-0004, Japan. E-mail: [email protected] Received 3 October 2011; revised 6 December 2011; accepted 5 January 2012; published online 2 February 2012 OFCD with BCOR mutation YKondoet al 198

heart defects in affected males, suggesting that genomic rearrangements of NHS are able to cause the X-linked cataract. In this report, mutation screening and genomic microarray revealed a heterozygous mutation in BCOR and genomic rearrangements involving NHS in the three affected females of a Korean family with congenital cataract, dental abnormalities and 2nd–3rd toe syndactyly. Detailed molecular analysis will be presented.

MATERIALS AND METHODS Clinical report The Korean family with congenital cataract was previously described (as family 4) (Figure 1a).12 Clinical features are summarized in Table 1. In the elder sister (MC17, the proband), bilateral congenital cataracts were noted 100 days after birth. Bilateral lensectomy and secondary intraocular lens insertion were performed. Ventricular septal defect, atrial septal defect, patent ductus arteriosus, delayed dentition, bilateral broad halluces, bilateral 2nd–3rd toe syndactyly, bilateral hammer toes and right brachyphalangia of fourth toe were also recognized. Mental development was normal. In the younger sister (MC18), bilateral congenital cataracts were also recognized. Bilateral lensectomy and secondary intraocular lens insertion were performed at ages of 2 months and 3 years, respectively. Right inguinal hernia, delayed dentition, and bilateral broad halluces, bilateral 2nd–3rd toe syndactyly, and bilateral hammer toes were noted (Figures 1b and c). She had learning difﬁculties at school, but IQ was not measured. In the mother (MC17b), bilateral congenital cataracts were noted and left lensectomy was performed at age of 10 years. Because of her dental anomalies and hypodontia, all her teeth were surgically removed. Bilateral 2nd–3rd toe syndactyly and bilateral hammer toes were noted. Her intelligence was normal. All the three affected members shared bilateral congenital cataracts, delayed dentition, bilateral 2nd–3rd toe syndactyly and bilateral hammer toes. Dysmorphic facial features were unseen.

DNA sequencing Experimental protocols were approved by Institutional Review Boards for Ethical Issues at Yokohama City University School of Medicine and the Committee for the Ethical Issues on Human Genome and Gene Analysis, Seoul National University. Informed consent was obtained from all individuals. Genomic DNA was obtained from peripheral leukocytes using QIAGEN Blood and Cell Culture DNA Midi Kit (QIAGEN, Hilden, Germany). DNA was Figure 1 Pedigree, foot malformation and a BCOR mutation found in the amplified using GenomiPhi V2 kit (GE healthcare, Buckinghamshire, UK). In family. (a) Familial pedigree. Black and open symbols denote affected and BCOR, there are three isoforms: isoform a (GenBank accession number unaffected individuals, respectively. (b, c) Bilateral broad halluces (the big toe, arrows in b), bilateral 2nd–3rd cutaneous syndactyly (arrowheads in c) NM_017745.5), isoform b (GenBank accession number NM_001123384.1) and bilateral hammer toes in MC18. (d) Schematic representation of the and isoform c (GenBank accession number NM_001123385.1). In NHS,there BCOR gene (top). UTR and coding region are open and filled rectangles, are two isoforms: isoform 1 (GenBank accession number NM_198270.2) and respectively. Alternative splicing by three different isoforms is shown. The isoform 2 (GenBank accession number NM_001136024.2). Nucleotide isoform b is absence of exon 5 and the isoform c is 102bp and 156bp sequences of 1st to 15th exons of BCOR and 1st to 8th exons of NHS covering longer than the isoform a and b, respectively. The location of the c.888delG all the protein coding region as well as exon–intron borders were analyzed. mutation is indicated by an arrow. The protein structure of BCOR (isoform a, Polymerase chain reaction (PCR) conditions and primer information are bottom). Three consecutive ankyrin motifs are indicated as a dark-gray box. shown in Supplementary Table 1. PCR products were purified with ExoSAP The three binding sites for BCL-6, AF9 and NSPC1 are indicated with (USB, Cleveland, OH, USA) and sequenced with BigDye terminator 3.1 horizontal bars. (e) Electropherograms showing the mutation in the affected (Applied Biosystems, Foster City, CA, USA) on 3100 and 3500Âl Genetic patient (MC17b) (top) and a control (bottom). A single nucleotide deletion in Analyzer (Applied Biosystems). Sequences of patients were compared with the exon 4 results in a frameshift. mut, a mutant allele; wt, a wild type allele. reference human genome sequences (based on the UCSC Genome Browser coordinate, February 2009) with Sequencher 4.10.1. (Gene Codes, Ann Arbor, MI, USA). encompassed exons and (3) CNVs were not present in the Database of Genomic Variants (http://projects.tcag.ca/variation/). Copy number analysis Copy number variations (CNVs) were investigated by Cytogenetics Whole- Genome 2.7 M. Array (Affymetrix, Santa Clara, CA, USA) based on the Cloning of duplication breakpoints manufacturer’s instruction using 100 ng genomic DNA from three affected DNA of the affected mother (MC17b) was digested with restriction enzymes: females (MC17b, MC17 and MC18). Copy number alterations were analyzed EcoRI, NsiI, XbaI, BamHI and BglII (New England Biolabs, Beverly, MA, USA). by Chromosome Analysis Suite (Affymetrix) with NetAffx 30.1 annotations Digested DNA was self-ligated by Ligation High ver. 2 (Toyobo, Osaka, Japan), (hg18 assembly). Any filters such as minimum size and probe numbers of precipitated with ethanol, and dissolved in 20 ml EB buffer (QIAGEN, Tokyo, CNVs were not applied. The selection criteria for putative pathogenic CNVs Japan). Inverse PCR was performed in 25 ml volume containing 2 ml ligated were as follows: (1) CNVs were shared with three affected females, (2) CNVs DNA, 1Â PCR buffer for KOD FX, 0.4 mM each dNTPs, 0.5 mM each primer and

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Table 1 Clinical features of patients with a BCOR mutation X chromosome haplotype was analyzed using 12 microsatellite markers (DXS1060, DXS8051, DXS987, DXS1226, DXS1214, DXS1068, DXS993, MC17b MC17 MC18 DXS991, DXS986, DXS8055, DXS1047 and DXS1073). Fluorescent- labeled (either FAM, VIC or NED) forward primers and tailed reverse primers were Age 41 11 8 purchased from Applied Biosystems. These markers were based on the Marsh- Sex Female Female Female ﬁeld genetic map (http://research.marshﬁeldclinic.org/genetics). PCR was cycled 40 times at 94 1C for 30 s, 55 1C for 30 s and 72 1C for 30 s in 10 ml Ocular features volume containing 50 ng DNA, 1Â ExTaq buffer, 0.2 mM each dNTP, 0.4 mM Congenital cataract + + + each primer and 0.1 U ExTaq HS polymerase. Haplotypes were manually Microphthalmia/microcornea ÀÀÀconstructed. Coloboma ÀÀÀ Ptosis ÀÀÀRESULTS Secondary glaucoma À ++We detected a BCOR mutation, c. 888delG in MC17, MC17b and Nystagmus ÀÀÀMC18 (Figures 1d and e). The mutation may result in insertion of 80 new amino acids after the mutation site with a premature stop codon Cardiac features À + À at position 377 (p.N297IfsX80). The mutation was completely Septal defects À + À co-segregated with OFCD phenotypes in this family (Figure 1a). Patent ductus arteriosus À + À Sequencing of the entire NHS coding region detected no pathological mutations in this family. Dental features The Cytogenetics Whole-Genome 2.7 M array detected a total of 48 Delayed/persistent/unerupted dentition + + + CNVs (12 duplications and 36 deletions) in any of the affected Root radiculomegaly (secondary teeth) ND ND ND females. The CNVs, which encompassed exons, were 8 duplications Hypodontia (secondary teeth) + ND ND and 13 deletions. Among them, two duplications and one deletion Duplication/fusion (secondary teeth) ND ND ND were shared with three affected females. The one deletion was present

Skeletal features in the Database of Genomic Variants. Thus, the CNVs fulfilling the Hammer toes (camptodactyly) + + + criteria for pathogenic CNVs were two interstitial duplications. The Second–third toe syndactyly + + + 740-kb duplication at Xp22.2–22.13 encompassed exons 2–18 of Broad halluces (big toe) À ++REPS2 (MIM *300317) and exons 1–3 of NHS. The other 110-kb Brachyphalangia of the fourth right toe À + À duplication at Xp21.3 contained exon 2 of interleukin-1 receptor Radioulnar synostosis ND ND ND accessory protein-like 1 (IL1RAPL1) (MIM *300206) (Figure 2a). We Lodosis/scoliosis/vertebral fusion ND ND ND were unable to examine the duplications by fluorescent in situ hybridization because only DNA samples were available. Instead, Other features inverse PCR of self-ligated DNA with different sets of primers was Mental retardation ÀÀ+ able to amplify an expected fragment from normal NHS allele in all Hearing impairment À ND ND sets of primers, suggesting that the presence of one or more normal Inguinal hernia ÀÀ+ NHS alleles (Figures 2b and c). In addition, several attempts success- Abbreviation: ND, Not determined. fully cloned one of rearrangement junctions in relation to NHS A plus (+) or minus (À) sign denotes the presence or absence of a particular physical feature. (Figure 2c). This aberrant band showed that the sequences of intron 1ofIL1RAPL1 followed the sequences of intron 3 of NHS,suggesting that two duplications were tandemly connected (Figure 2d, upper 0.5 U KOD FX polymerase (Toyobo). PCR were cycled 35 times at 98 1Cfor cases). More complicatedly, 62-bp sequences of intron 3 of IL1RAPL1 10 s, 68 1C for 10 min. PCR products electrophoresed in 1% agarose gel were with inverted orientation were inserted between intron 3 of NHS and stained with ethidium bromide and the aberrant band was extracted using intron 1 of IL1RAPL1 (Figure 2d, lower cases). The other possible QIAEXII Gel Extraction Kit (QIAGEN, Tokyo, Japan) and sequenced. Primer breakpoints, which may result in disruption of NHS locus could not information is available on request. be determined regardless of rigorous attempts. Thus there seems to be two normal NHS alleles and an extra NHS allele, in which exons 1–3 X inactivation study and haplotype analysis of NHS was connected to exon 2 of IL1RAPL1.Copynumberofthe X inactivation pattern was studied using the human androgen receptor assay BCOR gene was normal. In human androgen receptor assays, the and fragile X mental retardation locus methylation assay as previously mother (MC17b) was skewed pattern (8%), whereas the elder sister described.13–15 Briefly, genomic DNA of a patient (MC17), the parents (MC17) showed a random pattern (26%). In fragile X mental (MC17a and MC17b), a control male and a control female was digested with retardation assays, the mother (MC17b) and elder sister (MC17) two methylation-sensitive enzymes, HpaII and HhaI. PCR was performed with showed a highly skewed pattern (o1%) and a random pattern human androgen receptor assay primers (FAM-labeled ARf: 5¢-CCAGAAT (48%), respectively. X-chromosome haplotype analysis was able to CTGTTCCAGAGCGTGC-3¢;ARr:5¢-CTCTACGATGGGCTTGGGGAGAA separate all the alleles in this family (Supplementary Figure 1). The 16 C-3¢) and fragile X mental retardation assay primers (FAM-labeled FMR1f: inactivated allele in the mother harbored the BCOR mutation and the ¢ ¢ ¢ 5 -AGCCCCGCACTTCCACCACCAGCTCCTCCA-3 ;FMR1r:5-GCTCAGCT NHS rearrangement. CCGTTTCGGTTTCACTTCCGGT-3¢). Fluorescent-labeled products were analyzed on an ABI PRISM 3100 or 3130Âl Genetic analyzer and GeneMapper DISCUSSION Software version 4.0 (Applied Biosystems). One of affected females (MC18) was not analyzed because of insufficient amount of genomic DNA. According to BCOR functions as a corepressor of BCL-6, which is a POZ/zinc finger 4 published criteria, X inactivation ratios of p80:20 were considered random transcription repressor. BCOR have three consecutive ankyrin motifs, and ratios 480:20 were considered skewed and ratios 490:10 were considered an AF9 (ALL1 fused gene from chromosome 9) binding site and an highly skewed.16,17 NSPC1 (nervous system polycomb-1) binding site.4,18,19 Recently, the

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explain the OFCD phenotypes because BCL-6-deficient mice did not show ocular, dental and skeletal phenotypes.21,22 Moreover, an OFCD mutant protein, that lacked ankyrin motifs and NSPC1 binding site, showed transcriptional repression activity similar to that of wild type.3 Thus, the c.888delG mutation in BCOR may be associated with loss of BCOR transcripts through nonsense-mediated mRNA decay. By genomic microarray, two microduplications were detected in MC17, MC18 and MC17b: one at Xp22.2–22.13 involving a part of REPS2 and NHS, and the other at Xp21.3 containing exon 2 of IL1RAPL1. Previously, complex duplication-triplication rearrangements of the NHS gene in a family with congenital cataract were reported, suggesting that genomic rearrangements of NHS are able to cause the X-linked cataract.8 This family did not show the typical features of NHS such as dental anomalies, dysmorphism and devel- opmental delay, and congenital heart defects were diagnosed in four out of six affected males. The complex rearrangement consists of triplication embedded within a duplication region. The triplicated region includes the NHS genes except exon 1, and the entire SCML1 and RAI2 genes. Coccia et al.8 described that the additional phenotype of congenital heart defects observed in some affected males could be because of perturbed NHS gene transcription or increased dosage of the NHS, SCML1 or RAI2 genes. In our cases, two normal NHS alleles may exist in addition to an extra NHS allele, in which exons 1–3 of NHS was connected to individual exon 2 of IL1RAPL1, keeping the protein coding frame if properly spliced. Because the transcript from the extra NHS allele did not have polyA signal sequences, the allele is likely to produce no functional protein. Thus, the situation was totally different between our cases and Coccia’s et al.’s 8 cases. Recently, Honda et al.23 reported two unrelated X-linked mental retardation Japanese families, which possessed the similar duplication found in our Korean family: the 737-kb duplication at Xp22.2, which Figure 2 Two microduplications at Xp22.2–22.13 and Xp21.3. (a) Array contains a part of REPS2 and NHS, and the 100-kb duplication at profile of a part of chromosome X in the elder sister (MC17). x and y axis Xp21.3, which contains a part of IL1RAPL1. In their report, fluor- show the genomic location from the p telomere of chromosome X (UCSC escent in situ hybridization analysis revealed that the clone RP11- coordinates, May, 2006) (upper) and log2 (Signal ratio) values (lower), 438J7, which entirely covered the duplication at Xp21.3, demonstrated respectively. The 740-kb duplication at Xp22.2–22.13 and the 110-kb two distinct signals at Xp in metaphases, suggesting that the duplica- duplication at Xp21.3 are indicated by arrows. (b) Upper shows schematic tion at Xp21.3 was inserted separately from the original site. The clone representation of two duplicated regions (highlighted in gray). Three genes (REPS2, NHS and IL1RAPL1) are oriented in the same direction (from RP11-2K15 at Xp22.2, spanning the breakpoint of REPS2,showedone centromere to telomere). Lower indicates scheme of inverse PCR using self- bright strong signal, suggesting that the duplication at Xp22.2 ligated DNA after digestion with EcoRI. Primers are shown by arrows (F, occurred in the proximity. Interestingly, the one of two signals of forward; R, reverse). (c) Gel image of inverse PCR products performed as the RP11-438J7 was close to that of RP11-2K15 at Xp22.2, suggesting shown in (b). Upper and lower bands represent wild-type allele (WT allele) that the duplication involving IL1RAPL1 was inserted at Xp22.2. Their and rearranged allele (Rea allele), respectively. The DNA of both bands was result is consistent with our data of the duplication breakpoint, in extracted from agarose gel and sequenced. (d) Sequence of a Rea allele is shown. The top, middle and bottom indicate sequences of the intron 3 of which the breakpoint in NHS was connected with the breakpoint of NHS, rearranged junction and intron 1 of IL1RAPL1, respectively. The 62 IL1RAPL1. Based on our experiences, high-density array experiments nucleotides of intron 3 of IL1RAPL1 (lower cases) were inserted in an of 4500 Japanese cases never showed such the genomic rearrange- inverted orientation between intron 3 of NHS and intron 1 of IL1RAPL1. ment involving NHS, implying that the rearrangement is very rare in Matched sequences are highlighted with gray shadows. Japanese population. It is noteworthy that congenital cataract and dental abnormalities were not pointed out in all the members (males and carrier females).23 Thus, it is unlikely that the genomic rearrange- minimal BCL-6 binding site was identified within residues 498–514, ment involving NHS causes congenital cataract and dental abnorm- which is located in exon 4.20 In this study, a novel BCOR mutation, alities as found in our family. c.888delG (p.N297IfsX80) in exon 4, was found in this family. We IL1RAPL1 is a causative gene for X-linked mental retardation,24 and assumed the mutant transcript with this mutation may undergo the microduplication at Xp21.3 containing exon 2 of IL1RAPL1 was nonsense-mediated mRNA decay, but we could not confirm it as no suggested to cause MR in affected males.23 Most carrier females of living cells were available from the patients. A total of 31 mutations in IL1RAPL1 mutations and the carrier mother of the microduplication BCOR were registered in Human Gene Mutation Database (http:// involving IL1RAPL1 showed normal intelligence.23–25 In this study, one www.biobase-international.com/). All the mutations result in prema- of the three affected females (MC18) had learning difficulties at school, ture stop codons. Exon 4 harbors 40% of mutations (12/30), leading which could be mild presentation of MR. As 18% of patients with to truncated proteins lacking BCL-6, AF9 and NSPC1 binding sites, BCOR mutations showed MR,6 it is reasonable that the BCOR muta- and ankyrin motifs if translated. Lack of BCL-6 binding site could not tion, rather than IL1RAPL1 rearrangement, causes mild MR in MC18.

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Skewed and random X-inactivation in the mother (MC17b) and the 7 Nance, W. E., Warburg, M., Bixler, D. & Helveston, E. M. Congenital X-linked cataract, elder daughter (MC17) was confirmed, respectively, in this family. In dental anomalies and brachymetacarpalia. Birth Defects Orig. Artic. Ser. 10, 285–291 (1974). OFCD, skewed X-inactivation with the preferential inactivation of the 8 Coccia, M., Brooks, S. P., Webb, T. R., Christodoulou, K., Wozniak, I. O., Murday, V. mutated allele were recognized in eight affected females (like the et al. X-linked cataract and Nance-Horan syndrome are allelic disorders. Hum. Mol. Genet. 18, 2643–2655 (2009). mother, MC17b), suggesting that the BCOR mutations may lead to a 9 Lewis, R. A., Nussbaum, R. L. & Stambolian, D. Mapping X-linked ophthalmic diseases. 3,26 selective disadvantage in blood leukocytes. However, it has been IV. Provisional assignment of the locus for X-linked congenital cataracts and micro- reported that the X-inactivation pattern in blood leukocytes are cornea (the Nance-Horan syndrome) to Xp22.2-p22.3. Ophthalmology. 97, 110–120; discussion 120–111 (1990). unable to determine the severity of disease phenotypes as X-inactiva- 10 Stambolian, D., Lewis, R. A., Buetow, K., Bond, A. & Nussbaum, R. Nance-Horan tion pattern may vary among tissues.27 We suspect that X-inactivation syndrome: localization within the region Xp21.1-Xp22.3 by linkage analysis. Am. pattern is different between peripheral blood leukocytes and respective J. Hum. Genet. 47, 13–19 (1990). 11 Burdon, K. P., McKay, J. D., Sale, M. M., Russell-Eggitt, I. M., Mackey, D. A., Wirth, M. tissues associated with OFCD phenotype. G. et al. Mutations in a novel gene, NHS, cause the pleiotropic effects of Nance-Horan In conclusion, a new OFCD family is described with a novel BCOR syndrome, including severe congenital cataract, dental anomalies, and mental retardation. Am.J.Hum.Genet.73, 1120–1130 (2003). mutation. Clinical features overlap between OFCD and NHS, both of 12 Miyamoto, T., Yu, Y. S., Sato, H., Hayashi, H., Sakugawa, N., Ishikawa, M. et al. 6 which belong to a spectrum of X-linked microphthalmia disorders. Mutational analysis of the human MBX gene in four Korean families demonstrating Our cases have a BCOR mutation and genomic rearrangements microphthalmia with congenital cataract. Turk J. Pediatr. 49, 334–336 (2007). 13 Allen, R. C., Zoghbi, H. Y., Moseley, A. B., Rosenblatt, H. M. & Belmont, J. W. involving NHS, confusing us to address the genetic etiology in the Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human family. However, the presence of bilateral 2nd–3rd toe syndactyly and androgen-receptor gene correlates with X chromosome inactivation. Am. J. Hum. Genet. septal heart defects, which is unique to OFCD, can lead us to the 51, 1229–1239 (1992). 14 Carrel, L. & Willard, H. F. An assay for X inactivation based on differential methylation conclusion that the BCOR mutation is the major determinant for the at the fragile X locus, FMR1. Am.J.Med.Genet.64, 27–30 (1996). phenotypes in this family. Careful examination of associated anoma- 15 Nishimura-Tadaki, A., Wada, T., Bano, G., Gough, K., Warner, J., Kosho, T. et al. Breakpoint determination of X; autosome balanced translocations in four patients with lies will be useful for genetic testing of X-linked microphthalmia premature ovarian failure. J. Hum. Genet. 56, 156–160 (2011). disorders. 16 Kubota, T., Nonoyama, S., Tonoki, H., Masuno, M., Imaizumi, K., Kojima, M. et al. A new assay for the analysis of X-chromosome inactivation based on methylation-specific PCR. Hum. Genet. 104, 49–55 (1999). ACKNOWLEDGEMENTS 17 Amos-Landgraf, J. M., Cottle, A., Plenge, R. M., Friez, M., Schwartz, C. E., Longshore, J. We thank the family members for their participation in this study. This work et al. X chromosome-inactivation patterns of 1,005 phenotypically unaffected was supported by Research Grants from the Ministry of Health, Labour and females. Am. J. Hum. Genet. 79, 493–499 (2006). 18 Srinivasan, R. S., de Erkenez, A. C. & Hemenway, C. S. The mixed lineage leukemia Welfare (HS, N Miyake and N Matsumoto) and the Japan Science and fusion partner AF9 binds specific isoforms of the BCL-6 corepressor. Oncogene 22, Technology Agency (N Matsumoto) and Grant-in-Aid for Scientific Research 3395–3406 (2003). from Japan Society for the Promotion of Science (N Matsumoto). 19 Gearhart, M. D., Corcoran, C. M., Wamstad, J. A. & Bardwell, V. J. 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